The organophosphate nerve agents tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), VX, and Russian VX (R-VX) are among the deadliest compounds known. Current treatments for chemical weapons exposure offer limited protection, must be administered immediately, and can generate long-term toxic side effects. Human carboxylesterase 1 (hCE1), a promiscuous drug metabolism enzyme, offers promise as a broad- spectrum protein-based therapeutic for nerve agent detoxification. We have determined several crystal structures of hCE1 in complex with live nerve agents. These preliminary results have indicated that the enzyme is both stereoselective and resistant to aging, and provides two putative nerve agent binding sites: the active site and a promiscuous surface binding site. The focus of this application is to convert hCE1 into a highly efficient nerve agent hydrolase using a range of targeted structural, biochemical and in vivo studies. A team of investigators at the University of North Carolina at Chapel Hill, St. Jude Children's Research Hospital, and the US Army Medical Research Institute of Chemical Defense has been assembled, and four specific aims will be pursued: 1. Elucidate crystal structures of hCE1 in complex with a wide variety of live nerve agents. 2. Introduce targeted mutations designed to improve nerve agent binding and hydrolysis. 3. Examine the impact site-directed mutants have on hCETs nerve agent hydrolysis activity in vitro. 4. Assess the in vivo ability of engineered forms of hCE1 to protect serum esterase-deficient mice from exposure to nerve agents. The overall goal of these on-going investigations is to develop a novel, efficient, broad-spectrum protein- based therapeutic for prophylactic protection against nerve agent exposure. It is clear that effective catalytic countermeasures are urgently needed to safely protect military personnel and civilian first-responders from attacks involving chemical weapons.